Tartrate salt of a substituded dipeptide as growth hormone secretagogue

ABSTRACT

This invention is directed to the (L)-tartaric acid salt of 2-amino-N-{1 -(R)-(2,4-difluoro-benzyloxymethyl)-2-oxo-2-[3-oxo-3a-(R)-pyridin-2-ylmethyl-2-(2,2,2-trifluoroethyl)-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5-yl]-ethyl}-2-methyl-propionamide which is a growth hormone secretagogue and as such is useful for increasing the level of endogenous growth hormone. In another aspect, this invention provides certain intermediates which are useful in the synthesis of the foregoing compound. The (L)-tartaric acid salt of the compound of this invention is useful for the treatment and/or prevention of osteoporosis, insulin resistance and other conditions or diseases associated with growth hormone deficiency. The (L)-tartaric acid salt of the compound of the compound of the present invention is also useful in treating osteoporosis when used in combination with: a bisphosphonate compound; estrogen, Premarin, and optionally progesterone; an estrogen agonist or antagonist; or calcitonin. Further, the present invention is directed to pharmaceutical compositions. This invention is further directed to methods comprising administering to a human or other animal a combination of an alpha-2 adrenergic agonist and the (L)-tartaric acid salt of the compound of this invention.

BACKGROUND OF THE INVENTION

[0001] This invention relates to the (L)-(+)-tartaric acid salt of2-amino-N-{1-(R)-(2,4-difluoro-benzyloxymethyl)-2-oxo-2-[3-oxo-3a-(R)-pyridin-2-ylmethyl-2-(2,2,2-trifluoro-ethyl)-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5-yl]-ethyl}-2-methyl-propionamidewhich is a growth hormone secretagogue.

[0002] Growth hormone (GH), which is secreted from the pituitary gland,stimulates growth of all tissues of the body that are capable ofgrowing. In addition, growth hormone is known to have the followingbasic effects on the metabolic processes of the body:

[0003] 1. Increased rate of protein synthesis in substantially all cellsof the body;

[0004] 2. Decreased rate of carbohydrate utilization in cells of thebody; and

[0005] 3. Increased mobilization of free fatty acids and use of fattyacids for energy.

[0006] Deficiency in growth hormone results in a variety of medicaldisorders. In children, it causes dwarfism. In adults, the consequencesof acquired GH deficiency include profound reduction in lean body massand concomitant increase in total body fat, particularly in the truncalregion. Decreased skeletal and cardiac muscle mass and muscle strengthlead to a significant reduction in exercise capacity. Bone density isalso reduced. Administration of exogenous growth hormone has been shownto reverse many of the metabolic changes. Additional benefits of therapyhave included reduction in LDL cholesterol and improved psychologicalwell-being.

[0007] In cases where increased levels of growth hormone were desired,the problem was generally solved by providing exogenous growth hormoneor by administering an agent which stimulated growth hormone productionand/or release. In either case the peptidyl nature of the compoundnecessitated that it be administered by injection. Initially the sourceof growth hormone was the extraction of the pituitary glands ofcadavers. This resulted in an expensive product, and carried with it therisk that a disease associated with the source of the pituitary glandcould be transmitted to the recipient of the growth hormone (e.g.,Jacob-Creutzfeld disease). Recently, recombinant growth hormone hasbecome available which, while no longer carrying any risk of diseasetransmission, is still a very expensive product which must be given byinjection or by a nasal spray.

[0008] Most GH deficiencies are caused by defects in GH release, notprimary defects in pituitary synthesis of GH. Therefore, an alternativestrategy for normalizing serum GH levels is by stimulating its releasefrom somatotrophs. Increasing GH secretion can be achieved bystimulating or inhibiting various neurotransmitter systems in the brainand hypothalamus. As a result, the development of synthetic growthhormone-releasing agents to stimulate pituitary GH secretion are beingpursued, and may have several advantages over expensive and inconvenientGH replacement therapy. By acting along physiologic regulatory pathways,the most desirable agents would stimulate pulsatile GH secretion, andexcessive levels of GH that have been associated with the undesirableside effects of exogenous GH administration would be avoided by virtueof intact negative feedback loops.

[0009] Physiologic and pharmacologic stimulators of GH secretion includearginine, L-3,4-dihydroxyphenylalanine (L-DOPA), glucagon, vasopressin,and insulin induced hypoglycemia, as well as activities such as sleepand exercise, indirectly cause growth hormone to be released from thepituitary by acting in some fashion on the hypothalamus perhaps eitherto decrease somatostatin secretion or to increase the secretion of theknown secretagogue growth hormone releasing factor (GHRF) or an unknownendogenous growth hormone-releasing hormone or all of these.

[0010] This invention also relates to a method of treating insulinresistant conditions such as Non-Insulin Dependent Diabetes (NIDD) andreduced glycemic control associated with obesity and aging in a mammalin need thereof which comprises administering to said mammal aneffective amount of the L-(+)-tartrate salt of the compound of FormulaI, shown below.

[0011] Other compounds have been developed which stimulate the releaseof endogenous growth hormone such as analogous peptidyl compoundsrelated to GRF or the peptides of U.S. Pat. No. 4,411,890. Thesepeptides, while considerably smaller than growth hormones are stillsusceptible to various proteases. As with most peptides, their potentialfor oral bioavailability is low.

[0012] WO 94/13696 refers to certain spiropiperidines and homologueswhich promote release of growth hormone. Preferred compounds are of thegeneral structure shown below.

[0013] WO 94/11012 refers to certain dipeptides that promote release ofgrowth hormone. These dipeptides have the general structure

[0014] where L is

[0015] The compounds of WO 94/11012 and WO 94/13696 are reported to beuseful in the treatment of osteoporosis in combination with parathyroidhormone or a bisphosphonate.

[0016] A generic disclosure of pharmaceutically-acceptable salts of thecompound of Formula I of the instant application is disclosed, and thefree base of the compound of Formula I of the instant invention isdisclosed and claimed, in co-pending PCT application Ser. No. PCT/IB96/01353 having an international filing date of Dec. 4, 1996, assignedto the assignee hereof.

[0017] It has been found that the L-(+)-tartaric acid salt of thecompound of Formula I, shown below, can be isolated in crystalline formwhich has advantageous properties such as ease of making a formulation,high solubility, good stability and is more easily purified than anon-crystalline form.

SUMMARY OF THE INVENTION

[0018] This invention provides the L-(+)-tartaric acid salt of thecompound of

[0019] are the stereochemical mixture or separated isomers having theconfigurations 3a-(S), 1-(R); 3a-(S), 1-(S); 3a-(R), 1-(S); and/or3a-(R), 1-(R) isomers.

[0020] This invention also provides: a process for the preparation ofthe (D)-tararic acid or the (L)-tartaric acid salt of the compound offormula (E),

[0021] which comprises reacting the compound of formula (D),

[0022] with (D)-tartarc acid or (L)-tartaric acid in about 8:1 to about9:1 mixture of acetone:water at a temperature between about 0° C. toroom temperature. Preferred of the foregoing process is where(D)-tartaric acid is reacted with the compound of formula (D) and thecompound of formula (E) has the R-configuration;

[0023] a process for the preparation of the compound of formula (J),

[0024] which comprises reacting the compound of formula (E),

[0025] with the compound of formula (X),

[0026] where Prt is an amine protecting group and X is OH,—O(C₁-C₄)alkyl or halo, in the presence of an organic base and a peptidecoupling reagent at a temperature between about −78° C. to about −20° C.Preferred of the immediately foregoing process is where the peptidecoupling reagent is 1-propane phosphonic acid cyclic anhydride and thecompound of formula X has the R-configuration and the compound offormula E has the R-configuration. Even more preferred is a where Prt istert-butyloxycarbonyl in the immediately foregoing process; and

[0027] a process for the preparation of the (L)-(+)-tartaric acid saltof the compound of formula I,

[0028] which comprises reacting the compound of formula (E),

[0029] with the compound of formula (X),

[0030] where Prt is an amine protecting group and X is OH,—O(C₁-C₄)alkyl or halo, in the presence of an organic base and a peptidecoupling reagent at a temperature between about −78° C. to about −20°C., to yield the compound of formula (J),

[0031] deprotecting the compound of formula (J) under appropriatedeprotecting conditions to yield the compound of formula (K),

[0032] reacting the compound of formula (K) with (L)-(+)-tartaric acidin a reaction inert solvent to yield the (L)-(+)-tartaric acid salt ofthe compound of formula I. Preferred of the immediately foregoingprocess is where Prt is tert-butoxycarbonyl, even more preferred of theimmediately foregoing process is where the peptide coupling reagent is1-propane phosphonic acid cyclic anhydride and the compound of formula Ihas the absolute and relative configuration 3a-(R), 1-(R).

[0033] In another aspect, this invention provides for:

[0034] the R,S-enantiomeric mixture, the R-enantiomner or theS-enantiomer of the compound of the formula

[0035] where the (D)-tartanic acid or the (L)-tartaric acid salt ispreferred;

[0036] the 3a-(R,S), 1-(R) diastereomeric mixture, the 3a-(R), 1-(R)diastereomer or the 3a-(S), 1-(R) diastereomer of the compound of theformula

[0037] where Prt is an amine protecting group selected from the groupconsisting of t-BOC, FMOC and CBZ; and

[0038] the R,S-enantiomeric mixture, the R-enantiomer or theS-enantiomer of the compound of the formula

[0039] the R,S-enantiomeric mixture, the R-enantiomer or theS-enantiomer of the compound of the formula

[0040] where X is OH, —O(C₁-C₄)alkyl or halo and Prt is an amineprotecting group; and where X is OH, Prt is BOC and the stereocenter isin the R-configuration is preferred.

[0041] In yet another aspect, this invention provides (where thecompound of formula (I) is shown above):

[0042] methods for increasing levels of endogenous growth hormone in ahuman or other animal which comprise administering to such human oranimal an effective amount of the (L)-(+)-tartaric acid salt of thecompound of formula I;

[0043] pharmaceutical compositions which comprise apharmaceutically-acceptable carrier and an amount of the(L)-(+)-tartaric acid salt of the compound of formula I;

[0044] pharmaceutical compositions useful for increasing the endogenousproduction or release of growth hormone in a human or other animal whichcomprise a pharmaceutically acceptable carrier, an effective amount ofthe (L)-(+)-tartaric acid salt of the compound of formula I according toclaim 1 and a growth hormone secretagogue selected from the groupconsisting of GHRP-6, Hexarelin, GHRP-1, growth hormone releasing factor(GRF), IGF-1, IGF-2 and B-HT920 or an analog thereof;

[0045] methods for treating or preventing osteoporosis which compriseadministering to a human or other animal in need of such treatment orprevention an amount of the (L)-(+)-tartaric acid salt of the compoundof formula I which is effective in treating or preventing osteoporosis;

[0046] methods for treating or preventing diseases or conditions whichmay be treated or prevented by growth hormone which compriseadministering to a human or other animal in need of such treatment orprevention an amount of the (L)-(+)-tartaric acid salt of the compoundof formula I which is effective in promoting release of endogenousgrowth hormone; preferred is a method wherein the disease or conditionis congestive heart failure, obesity or frailty associated with aging;also preferred is a method wherein the disease or condition iscongestive heart failure; further preferred is a method wherein thedisease or condition is frailty associated with aging;

[0047] methods for accelerating bone fracture repair, attenuatingprotein catabolic response after a major operation, reducing cachexiaand protein loss due to chronic illness, accelerating wound healing, oraccelerating the recovery of bum patients or patients having undergonemajor surgery, which methods comprise administering to a mammal in needof such treatment an amount of the (L)-(+)-tartaric acid salt of thecompound of formula I which is effective in promoting release ofendogenous growth hormone; preferred is a method wherein the method isfor accelerating the recovery of patients having undergone majorsurgery; also preferred is a method wherein the method is foraccelerating bone fracture repair;

[0048] methods for improving muscle strength, mobility, maintenance ofskin thickness, metabolic homeostasis or renal homeostasis, which methodcomprise administering to a human or other animal in need of suchtreatment an amount of the (L)-(+)-tartaric acid salt of the compound offormula I which is effective in promoting release of endogenous growthhormone;

[0049] methods for the treatment or prevention of osteoporosis whichcomprise administering to a human or other animal with osteoporosiseffective amounts of a bisphosphonate compound and the (L)-(+)-tartaricacid salt of the compound of formula I; preferred of a method for thetreatment of osteoporosis is where the bisphosphonate compound isibandronate; preferred of the method for the treatment of osteoporosisis where the bisphosphonate compound is alendronate;

[0050] methods for the treatment or prevention of osteoporosis whichcomprise administering to a human or other animal with osteoporosiseffective amounts of estrogen or Premarin® and the (L)-(+)-tartaric acidsalt of the compound of formula I and, optionally, progesterone.

[0051] methods for the treatment of osteoporosis which compriseadministering to a human or other animal with osteoporosis effectiveamounts of calcitonin and the (L)-(+)-tartaric acid salt of the compoundof formula I;

[0052] methods to increase IGF-1 levels in a human or other animaldeficient in IGF-1 which comprise administering to a human or otheranimal with IGF-1 deficiency an effective amount of the (L)-(+)-tartaricacid salt of the compound of formula I;

[0053] methods for the treatment of osteoporosis which compriseadministering to a human or other animal with osteoporosis effectiveamounts of an estrogen agonist or antagonist and the of the(L)-(+)-tartaric acid salt of the compound of formula I; preferred is amethod wherein the estrogen agonist or antagonist is tamoxifen,droloxifene, raloxifene or idoxifene; also preferred is a method wherethe estrogen agonist or antagonist iscis-6(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalene-2-ol;(-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalene-2-ol;

[0054]cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalene-2-ol;cis-1-[6′-pyrrolodinoethoxy-3′-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahydro-naphthalene;1-(4′-pyrrolidinoethoxyphenyl)-2-(4″-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;cis-6-(4-hydroxyphenyl)-5-[4(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalene-2-ol;or1-(4′-pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydro-isoquinoline;

[0055] methods for increasing muscle mass, which methods compriseadministering to a human or other animal in need of such treatment aneffective amount of the (L)-(+)-tartaric acid salt of the compound offormula I;

[0056] methods for promoting growth in growth hormone deficient childrenwhich comprise administering to a growth hormone deficient child aneffective amount of the (L)-(+)-tartaric acid salt of the compound offormula I;

[0057] methods for treating insulin resistance in a mammal, whichcomprise administering to said mammal an effective amount of the(L)-(+)-tartaric acid salt of the compound of formula I; preferred is amethod where the condition associated with insulin resistance is type Idiabetes, type II diabetes, hyperglycemia, impaired glucose tolerance oran insulin resistant syndrome; also preferred is a method where thecondition associated with insulin resistance is associated with obesityor old age;

[0058] methods for increasing levels of endogenous growth hormone, whichcomprise administering to a human or other animal in need thereofeffective amounts of a functional somatostatin antagonist and the(L)-(+)-tartaric acid salt of the compound of formula I; preferred is amethod where the functional somatostatin antagonist is an alpha-2adrenergic agonist; and

[0059] methods of treating or preventing congestive heart failure,obesity or frailty associated with aging, which comprise administeringto a human or other animal in need thereof effective amounts of afunctional somatostatin antagonist and the of the (L)-(+)-tartaric acidsalt of the compound of formula I.

[0060] The instant compound of formula I promotes the release of growthhormone, is stable under various physiological conditions and may beadministered parenterally, nasally or by the oral route.

DETAILED DESCRIPTION OF THE INVENTION

[0061] The (L)-(+)-tartrate salt of the compound of Formula I can bemade by the following processes which includes processes known in thechemical arts for the production of compounds. Certain processes for themanufacture of the L-(+)-tartaric acid salt of the compound of Formula Iare provided as further features of the invention and are illustrated bythe reaction scheme, shown below.

[0062] The compound of the instant invention has the absolute andrelative configuration shown below:

[0063] which is designated as the 3a-(R), 1-(R) configuration. It can beprepared by the method described hereinbelow.

[0064] The growth hormone releasing (L)-(+)-tartaric acid salt of thecompound of Formula I is useful in vitro as a unique tool forunderstanding how growth hormone secretion is regulated at the pituitarylevel. This includes use in the evaluation of many factors thought orknown to influence growth hormone secretion such as age, sex,nutritional factors, glucose, amino acids, fatty acids, as well asfasting and non-fasting states. In addition, the (L)-(+)-tartaric acidsalt of the compound of Formula I can be used in the evaluation of howother hormones modify growth hormone releasing activity. For example, ithas already been established that somatostatin inhibits growth hormonerelease.

[0065] The (L)-(+)-tartaric acid salt of the compound of Formula I canbe administered to animals, including humans, to release growth hormonein vivo. The (L)-(+)-tartaric acid salt of the compound of Formula I isuseful for treatment of symptoms related to GH deficiency; to stimulategrowth or enhance feed efficiency of animals raised for meat productionto improve carcass quality; to increase milk production in dairy cattle;for improvement of bone or wound healing and for improvement in vitalorgan function. The (L)-(+)-tartaric acid salt of the compound ofFormula I by inducing endogenous GH secretion, will alter bodycomposition and modify other GH-dependent metabolic, immunologic ordevelopmental processes. For example, the compounds of the presentinvention can be given to chickens, turkeys, livestock animals (such assheep, pigs, horses, cattle, etc.), companion animals (e.g., dogs) ormay have utility in aquaculture to accelerate growth and improve theprotein/fat ratio in fish. In addition, the (L)-(+)-tartaric acid saltof the compound of Formula I can be administered to humans in vivo as adiagnostic tool to directly determine whether the pituitary is capableof releasing growth hormone. For example, the (L)-(+)-tartaric acid saltof the compound of Formula I can be administered in vivo to children.Serum samples taken before and after such administration can be assayedfor growth hormone. Comparison of the amounts of growth hormone in eachof these samples would be a means for directly determining the abilityof the patient's pituitary to release growth hormone.

[0066] Accordingly, the present invention includes within its scopepharmaceutical compositions comprising, as an active ingredient, the(L)-(+)-tartaric acid salt of the compound of Formula I in associationwith a pharmaceutically acceptable carrier. Optionally, thepharmaceutical compositions can further comprise an anabolic agent inaddition to the (L)-(+)-tartaric acid salt of the compound of Formula Ior another compound which exhibits a different activity, e.g., anantibiotic growth permittant or an agent to treat osteoporosis or withother pharmaceutically active materials wherein the combination enhancesefficacy and minimizes side effects.

[0067] Growth promoting and anabolic agents are well known in the artand include, but are not limited to, TRH, PTH, diethylstilbesterol,estrogens, β-agonists, theophylline, anabolic steroids, enkephalins, Eseries prostaglandins, compounds disclosed in U.S. Pat. No. 3,239,345,the disclosure of which is hereby incorporated by reference, e.g.,zeranol, compounds disclosed in U.S. Pat. No. 4,036,979, the disclosureof which is hereby incorporated by reference, e.g., sulbenox, andpeptides disclosed in U.S. Pat. No. 4,411,890, the disclosure of whichis hereby incorporated by reference.

[0068] The (L)-(+)-tartaric acid salt of the compound of Formula I incombination with other growth hormone secretagogues such as the growthhormone releasing peptides GHRP-6 and GHRP-1 as described in U.S. Pat.No. 4,411,890, the disclosure of which is hereby incorporated byreference, and publications WO 89/07110, WO 89/07111 and B-HT920 as wellas hexarelin and the newly discovered GHRP-2 as described in WO 93/04081or growth hormone releasing hormone (GHRH, also designated GRF) and itsanalogs or growth hormone and its analogs or somatomedins includingIGF-1 and IGF-2 or adrenergic agonists such as clonidine, xylazine,detomidine and medetomidine (clonidine, which is disclosed in U.S. Pat.No. 3,202,660 the disclosure of which is hereby incorporated byreference, xylazine, which is disclosed in U.S. Pat. No. 3,235,550 thedisclosure of which is hereby incorporated by reference andmedetomidine, which is disclosed in U.S. Pat. No. 4,544,664 thedisclosure of which is hereby incorporated by reference) or serotonin5HTID agonists such as sumitriptan or agents which inhibit somatostatinor its release such as physostigmine and pyridostigmine, are useful forincreasing the endogenous levels of GH in mammals. The combination ofthe (L)-(+)-tartaric acid salt of the compound of Formula I with GRFresults in synergistic increases of endogenous growth hormone.

[0069] As is well known to those skilled in the art, the known andpotential uses of growth hormone are varied and multitudinous [See“Human Growth Hormone”, Strobel and Thomas, Pharmacological Reviews, 46,pg. 1-34 (1994); T. Rosen et al., Horm Res, 1995; 43: pp. 93-99; M.Degerblad et al., European Journal of Endocrinology, 1995, 133: pp.180-188; J. O. Jorgensen, European Journal of Endocrinology, 1994, 130:pp. 224-228; K. C. Copeland et al., Journal of Clinical Endocrinologyand Metabolism, Vol. 78 No. 5, pp. 1040-1047; J. A. Aloi et al., Journalof Clinical Endocrinology and Metabolism, Vol. 79 No. 4, pp. 943-949; F.Cordido et al., Metab. Clin. Exp., (1995), 44(6), pp. 745-748; K. M.Fairhall et al., J. Endocrinol., (1995), 145(3), pp. 417-426; R. M.Frieboes et al., Neuroendocrinology, (1995), 61(5), pp. 584-589; and M.Llovera et al., Int. J. Cancer, (1995), 61(1), pp. 138-141]. Thus, theadministration of the (L)-(+)-tanaric acid salt of the compound ofFormula I for purposes of stimulating the release of endogenous growthhormone can have the same effects or uses as growth hormone itself.These varied uses of growth hormone may be summarized as follows:stimulating growth hormone release in elderly humans; treating growthhormone deficient adults; preventing catabolic side effects ofglucocorticoids; treating osteoporosis; stimulating the immune system;accelerating wound healing; accelerating bone fracture repair; treatinggrowth retardation; treating congestive heart failure as disclosed inPCT publications WO 95/28173 and WO 95/28174 (an example of a method forassaying growth hormone secretagogues for efficacy in treatingcongestive heart failure is disclosed in R. Yang et al., Circulation,Vol. 92, No. 2, p. 262, 1995); treating acute or chronic renal failureor insufficiency; treating physiological short stature, including growthhormone deficient children; treating short stature associated withchronic illness; treating obesity; treating growth retardationassociated with Prader-Willi syndrome and Tumer's syndrome; acceleratingthe recovery and reducing hospitalization of bum patients or followingmajor surgery such as gastrointestinal surgery; treating intrauterinegrowth retardation, skeletal dysplasia, hypercortisonism and Cushingssyndrome; replacing growth hormone in stressed patients; treatingosteochondrodysplasias, Noonans syndrome, sleep disorders, Alzheimer'sdisease, delayed wound healing, and psychosocial deprivation; treatingof pulmonary dysfunction and ventilator dependency; attenuating proteincatabolic response after a major operation; treating malabsorptionsyndromes; reducing cachexia and protein loss due to chronic illnesssuch as cancer or AIDS; accelerating weight gain and protein accretionin patients on TPN (total parenteral nutrition); treatinghyperinsulinemia including nesidioblastosis; adjuvant treatment forovulation induction and to prevent and treat gastric and duodenalulcers; stimulating thymic development and preventing age-relateddecline of thymic function; adjunctive therapy for patients on chronichemodialysis; treating immunosuppressed patients and enhancing antibodyresponse following vaccination; improving muscle strength, increasingmuscle mass, mobility, maintenance of skin thickness, metabolichomeostasis, renal homeostasis in the frail elderly; stimulatingosteoblasts, bone remodeling, and cartilage growth; treatingneurological diseases such as peripheral and drug induced neuropathy,Guillian-Barre Syndrome, amyotrophic lateral sclerosis, multiplesclerosis, cerebrovascular accidents and demyelinating diseases;stimulating the immune system in companion animals and treatingdisorders of aging in companion animals; growth promotant in livestock;and stimulating wool growth in sheep.

[0070] It will be known to those skilled in the art that there arenumerous compounds now being used in an effort to treat the diseases ortherapeutic indications enumerated above. Combinations of thesetherapeutic agents, some of which have also been mentioned above, withthe growth promotant, exhibit anabolic and desirable properties of thesevarious therapeutic agents. In these combinations, the therapeuticagents and the (L)-(+)-tartaric acid salt of the compound of Formula Imay be independently and sequentially administered or co-administered indose ranges from one one-hundredth to one times the dose levels whichare effective when these compounds and secretagogues are used singly.Combined therapy to inhibit bone resorption, prevent osteoporosis,reduce skeletal fracture, enhance the healing of bone fractures,stimulate bone formation and increase bone mineral density can beeffectuated by combinations of bisphosphonates and the (L)-(+)-tartaricacid salt of the compound of Formula I. See PCT publication WO 95/11029for a discussion of combination therapy using bisphosphonates and GHsecretagogues. The use of bisphosphonates for these utilities has beenreviewed, for example, by Hamdy, N. A. T., Role of Bisphosphonates inMetabolic Bone Diseases, Trends in Endocrinol. Metab., 1993, 4, pages19-25. Bisphosphonates with these utilities include but are not limitedto alendronate, tiludronate, dimethyl-APD, risedronate, etidronate,YM-175, clodronate, pamidronate, and BM-210995 (ibandronate). Accordingto their potency, oral daily dosage levels of the bisphosphonate ofbetween 0.1 mg and 5 g and daily dosage levels of the (L)-(+)-tartaricacid salt of the compound of Formula I of between 0.01 mg/kg to 20 mg/kgof body weight are administered to patients to obtain effectivetreatment of osteoporosis.

[0071] The (L)-(+)-tartaric acid salt of the compound of Formula I maybe combined with a mammalian estrogen agonist/antagonist. Any estrogenagonist/antagonist may be used as the second compound of this aspect ofthis invention. The term estrogen agonist/antagonist refers to compoundswhich bind with the estrogen receptor, inhibit bone turnover and preventbone loss. In particular, estrogen agonists are herein defined aschemical compounds capable of binding to the estrogen receptor sites inmammalian tissue, and mimicking the actions of estrogen in one or moretissue. Estrogen antagonists are herein defined as chemical compoundscapable of binding to the estrogen receptor sites in mammalian tissue,and blocking the actions of estrogen in one or more tissues. Suchactivities are readily determined by those skilled in the art accordingto standard assays including estrogen receptor binding assays, standardbone histomorphometric and densitometer methods (see Eriksen E. F. etal., Bone Histomorphometry, Raven Press, New York, 1994, pages 1-74;Grier S. J. et. al., The Use of Dual-Energy X-Ray Absorptionmetry InAnimals, Inv. Radiol., 1996, 31(1):50-62; Wahner H. W. and Fogelman I.,The Evaluation of Osteoporosis- Dual Energy X-Ray Absorptionmetry inClinical Practice., Martin Dunitz Ltd., London 1994, pages 1-296). Avariety of these compounds are described and referenced below, however,other estrogen agonistslantagonists will be known to those skilled inthe art. A preferred estrogen agonist/antagonist is droloxifene:(phenol, 3-[1-[4[2-(dimethylamino)ethoxy]-phenyl]-2-phenyl-1-butenyl]-,(E)-) and associated compounds which are disclosed in U.S. Pat. No.5,047,43, the disclosure of which is hereby incorporated by reference.

[0072] Another preferred estrogen agonist/antagonist is tamoxifen:(ethanamine,2-[-4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethyl, (Z)-2-,2-hydroxy-1,2,3-propanetri-carboxylate (1:1)) and associated compoundswhich are disclosed in U.S. Pat. No. 4,536,516, the disclosure of whichis hereby incorporated by reference. Another related compound is4-hydroxy tamoxifen which is disclosed in U.S. Pat. No. 4,623,660, thedisclosure of which is hereby incorporated by reference.

[0073] Another preferred estrogen agonist/antagonist is raloxifene:(methanone,[6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thien-3-yl][4-[2-(1-piperidinyl)ethoxy]phenyl]-,hydrochloride)and associated compounds which are disclosed in U.S. Pat. No. 4,418,068,the disclosure of which is hereby incorporated by reference.

[0074] Another preferred estrogen agonist/antagonist is idoxifene:pyrrolidine, 1-[-[4-[[1-(4-iodophenyl)-2-phenyl-1-butenyl]phenoxy]ethyl]and associated compounds which are disclosed in U.S. Pat. No. 4,839,155,the disclosure of which is hereby incorporated by reference.

[0075] Other preferred estrogen agonistlantagonists include compounds asdescribed in commonly assigned U.S. Pat. No. 5,552,412, the disclosureof which is hereby incorporated by reference. Especially preferredcompounds which are described therein are:

[0076]cis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalene-2-ol;

[0077] (-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalene-2-ol;

[0078]cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalene-2-ol;

[0079]cis-1-[6′-pyrrolodinoethoxy-3′-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahydronaphthalene;

[0080]1-(4′-pyrrolidinoethoxyphenyl)-2-(4″-fluorophenyl)6-hydroxy-1,2,3,4-tetrahydroisoquinoline;

[0081]cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalene-2-ol;and

[0082]1-(4′-pyrrolidinotethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroisoquinoline.

[0083] Other estrogen agonist/antagonists are described in U.S. Pat. No.4,133,814 (the disclosure of which is hereby incorporated by reference).U.S. Pat. No. 4,133,814 discloses derivatives of2-phenyl-3-aroyl-benzothiophene and2-phenyl-3-aroylbenzothiophene-1-oxide.

[0084] The following paragraphs provide preferred dosage ranges forvarious anti-resorptive agents.

[0085] The amount of the anti-resorptive agent to be used is determinedby its activity as a bone loss inhibiting agent. This activity isdetermined by means of an individual compound's pharmacokinetics and itsminimal maximal effective dose in inhibition of bone loss using aprotocol such as those referenced above.

[0086] In general an effective dosage for the activities of thisinvention, for example the treatment of osteoporosis, for the estrogenagonists/antagonists (when used in combination with (L)-(+)-tartaricacid salt of the compound of Formula I of this invention) is in therange of 0.01 to 200 mg/kg/day, preferably 0.5 to 100 mg/kg/day.

[0087] In particular, an effective dosage for droloxifene is in therange of 0.1 to 40 mg/kg/day, preferably 0.1 to 5 mg/kg/day.

[0088] In particular, an effective dosage for raloxifene is in the rangeof 0.1 to 100 mg/kg/day, preferably 0.1 to 10 mg/kg/day.

[0089] In particular, an effective dosage for tamoxifen is in the rangeof 0.1 to 100 mg/kg/day, preferably 0.1 to 5 mg/kg/day.

[0090] In particular, an effective dosage for

[0091]cis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalene-2-ol;

[0092](-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalene-2-ol;

[0093]cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalene-2-ol;

[0094]cis-1-[6′-pyrrolodinoethoxy-3′-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahydronaphthalene;

[0095]1-(4′-pyrrolidinoethoxyphenyl)-2-(4′-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;

[0096]cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalene-2-ol;or

[0097]1-(4′-pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroisoquinolineis in the range of 0.0001 to 100 mg/kg/day, preferably 0.001 to 10mg/kg/day.

[0098] In particular, an effective dosage for 4-hydroxy tamoxifen is inthe range of 0.0001 to 100 mg/kg/day, preferably 0.001 to 10 mg/kg/day.

[0099] Assay for Stimulation of GH Release From Rat Pituicytes

[0100] Compounds that have the ability to stimulate GH secretion fromcultured rat pituitary cells are identified using the followingprotocol. This test is also useful for comparison to standards todetermine dosage levels. Cells are isolated from pituitaries of 6-weekold male Wistar rats. Following decapitation, the anterior pituitarylobes are removed into cold, sterile Hank's balanced salt solutionwithout calcium or magnesium (HBSS). Tissues are finely minced, thensubjected to two cycles of mechanically assisted enzymatic dispersionusing 10 U/mL bacterial protease (EC 3.4.24.4, Sigma P-6141, St. Louis,Mo.) in HBSS. The tissue-enzyme mixture is stirred in a spinner flask at30 rpm in a 5% CO₂ atmosphere at about 37° C. for about 30 min., withmanual trituration after about 15 min. and about 30 min. using a 10 mLpipet. This mixture is centrifuged at 200 x g for about 5 min. Horseserum (35% final concentration) is added to the supematant to neutralizeexcess protease. The pellet is resuspended in fresh protease (10 U/mL),stirred for about 30 min. more under the previous conditions, andmanually triturated, ultimately through a 23-gauge needle. Again, horseserum (35% final concentration) is added, then the cells from bothdigests are combined, pelleted (200 x g for about 15 min.), resuspendedin culture medium (Dulbecco's Modified Eagle Medium (D-MEM) supplementedwith 4.5 g/L glucose, 10% horse serum, 2.5% fetal bovine serum, 1%non-essential amino acids, 100 U/mL nystatin and 50 mg/mL gentamycinsulfate, Gibco, Grand Island, N.Y.) and counted. Cells are plated at6.0-6.5×10⁴ cells per cm² in 0.48-well Costar™ (Cambridge, Mass.) dishesand cultured for 3-4 days in culture medium.

[0101] Just prior to GH secretion assay, culture wells are rinsed twicewith release medium, then equilibrated for about 30 minutes in releasemedium (D-MEM buffered with 25 mM Hepes, pH 7.4 and containing 0.5%bovine serum albumin at 37° C.). Test compounds are dissolved in DMSO,then diluted into pre-warmed release medium. Assays are run inquadruplicate. The assay is initiated by adding 0.5 mL of release medium(with vehicle or test compound) to each culture well. Incubation iscarried out at about 37° C. for about 15 minutes, then terminated byremoval of the release medium, which is centrifuged at 2000 x g forabout 15 minutes to remove cellular material. Rat growth hormoneconcentrations in the supernatants are determined by a standardradioimmunoassay protocol described below.

[0102] Measurement of Rat Growth Hormone

[0103] Rat growth hormone concentrations were determined by doubleantibody radioimmunoassay using a rat growth hormone referencepreparation (NIDDK-rGH-RP-2) and rat growth hormone antiserum raised inmonkey (NIDDK-anti-rGH-S-5) obtained from Dr. A. Pariow (Harbor-UCLAMedical Center, Torrence, Calif.). Additional rat growth hormone (1.5U/mg, #G2414, Scripps Labs, San Diego, Calif.) is iodinated to aspecific activity of approximately 30 μCi/μg by the chloramine T methodfor use as tracer. Immune complexes are obtained by adding goatantiserum to monkey IgG (ICN/Cappel, Aurora, Ohio) plus polyethyleneglycol, MW 10,000-20,000 to a final concentration of 4.3%; recovery isaccomplished by 30 centrifugation. This assay has a working range of0.08-2.5 μg rat growth hormone per tube above basal levels.

[0104] Assay for Exocenously-Stimulated Growth Hormone Release in theRat After Intravenous Administration of Test Compounds

[0105] Twenty-one day old female Sprague-Dawley rats (Charles RiverLaboratory, Wilmington, Mass.) are allowed to acclimate to localvivarium conditions 24° C., 12 hr light, 12 hr dark cycle) forapproximately 1 week before compound testing. All rats are allowedaccess to water and a pelleted commercial diet (Agway Country Food,Syracuse N.Y.) ad libitum. The experiments are conducted in accordancewith the NIH Guide for the Care and Use of Laboratory Animals.

[0106] On the day of the experiment, test compounds are dissolved invehicle containing 1% ethanol, 1 mM acetic acid and 0.1% bovine serumalbumin in saline. Each test is conducted in three rats. Rats areweighed and anesthetized via intrapertoneal injection of sodiumpentobarbital (Nembutol®, 50 mg/kg body weight). Fourteen minutes afteranesthetic administration, a blood sample is taken by nicking the tip ofthe tail and allowing the blood to drip into a microcentrifuge tube(baseline blood sample, approximately 100 μl). Fifteen minutes afteranesthetic administration, test compound is delivered by intravenousinjection into the tail vein, with a total injection volume of 1 mL/kgbody weight. Additional blood samples are taken from the tail at 5, 10and 15 minutes after compound administration. Blood samples are kept onice until serum separation by centrifugation (1430 x g for 10 minutes at10° C.). Serum is stored at −80° C. until serum growth hormonedetermination by radioimmunoassay as described above.

[0107] Assessment of Exogenously-Stimulated Growth Hormone Release inthe Dog After Oral Administration

[0108] On the day of dosing, the test compound is weighed out for theappropriate dose and dissolved in water. Doses are delivered at a volumeof 0.5-3 mL/kg by gavage to 2-4 dogs for each dosing regimen. Bloodsamples (5 mL) are collected from the jugular vein by direct venapuncture pre-dose and at 0.17, 0.33, 0.5, 0.75, 1, 2, 4, 6, 8 and 24hours post dose using 5 mL vacutainers containing lithium heparin. Theprepared plasma is stored at −20° C. until analysis.

[0109] Measurement of Canine Growth Hormone

[0110] Canine growth hormone concentrations are determined by a standardradioimmunoassay protocol using canine growth hormone (antigen foriodination and reference preparation AFP-1983B) and canine growthhormone antiserum raised in monkey (AFP-21452578) obtained from Dr. A.Pardow (Harbor-UCLA Medical Center, Torrence, Calif.). Tracer isproduced by chloramine T-iodination of canine growth hormone to aspecific activity of 20-40 μCi/μg. Immune complexes are obtained byadding goat antiserum to monkey IgG (ICN/Cappel, Aurora, Ohio) pluspolyethylene glycol, MW 10,000-20,000 to a final concentration of 4.3%;recovery is accomplished by centrifugation. This assay has a workingrange of 0.08-2.5 μg canine GH/tube.

[0111] Assessment of Canine Growth Hormone and Insulin-Like GrowthFactor-1 Levels in the Dog after Chronic Oral Administration

[0112] The dogs receive test compound daily for either 7 or 14 days.Each day of dosing, the test compound is weighed out for the appropriatedose and dissolved in water. Doses are delivered at a volume of 0.5-3ml/kg by gavage to 5 dogs for each dosing regimen. Blood samples arecollected at days 0, 3, 7, 10 and 14. Blood samples (5 ml) are obtainedby direct venipuncture of the jugular vein at pre-dose, 0.17, 0.33, 0.5,0.754, 1, 2, 3, 6, 8, 12 and 24 hours post administration on days 0, 7and 14 using 5 ml vacutainers containing lithium heparin. In addition,blood is drawn pre-dose and 8 hours on days 3 and 10. The preparedplasma is stored at −20° C. until analysis.

[0113] Female Rat Study

[0114] This study evaluates the effect of chronic treatment with a GHRPmimetic on weight, body composition and non-fasting plasmaconcentrations of glucose, insulin, lactate and lipids inestrogen-deficient and estrogen-replete female rats. Acuteresponsiveness of serum GH levels to i.v. administration of the GHreleasing agent was assessed on the last day of dosing. Body weight wasmonitored weekly throughout the treatment period; additionally, bodycomposition and plasma levels of glucose, insulin, lactate, cholesteroland triglycerides were assessed at the end of treatment.

[0115] Virgin female Sprague-Dawley rats were obtained from CharlesRiver Laboratories (Wilmington, Mass.) and underwent bilateralovariectomy (Ovx) or sham-surgery (Sham) at approximately 12 weeks ofage. For sham surgeries, ovaries were exteriorized and replaced into theabdominal cavity. Following surgery the rats were housed individually in20 cm×32 cm×20 cm cages under standard vivarium conditions (about 24° C.with about 12 hours light/12 hours dark cycle). All rats were allowedfree access to water and a pelleted commercial diet (Agway ProLab 3000,Agway Country Food, Inc., Syracuse, N.Y.). The experiment was conductedin accordance with NIH Guidelines for the Care and Use of laboratoryAnimals.

[0116] Approximately seven months post-surgery, Sham and Ovx rats wereweighed and randomly assigned to groups. Rats were dosed daily by oralgavage with 1 mL of either vehicle (1% ethanol in distilled-deionizedwater), 0.5 mg/kg or 5 mg/kg of a growth hormone releasing agent for 90days. Rats were weighed at weekly intervals throughout the study.Twenty-four hours after the last oral dose, the acute response of serumgrowth hormone (GH) to test agent was assessed by the followingprocedure. Rats were anesthetized with sodium pentobarbital 50 mg/kg.Anesthetized rats were weighed and a baseline blood sample (˜100 μl) wascollected from the tail vein. Test agent (growth hormone releasing agentor vehicle) was then administered intravenously via the tail vein in 1mL. Approximately ten minutes after injection, a second 100 μl bloodsample was collected from the tail. Blood was allowed to clot at about4° C., then centrifuged at 2000 x g for about 10 minutes. Serum wasstored at about −70° C. Serum growth hormone concentrations weredetermined by radioimmunoassay as previously described. Following thisprocedure, each anesthetized rat underwent whole body scanning bydual-energy X-ray absorptiometry (DEXA, Hiologic QDR 1000/W, WalthamMass.). A final blood sample was collected by cardiac puncture intoheparinized tubes. Plasma was separated by centrifugation and storedfrozen as described above.

[0117] Plasma insulin is determined by radioimmunoassay using a kit fromBinax Corp. (Portland, Me.). The interassay coefficient of variation is≦10%. Plasma triglycerides, total cholesterol, glucose and lactatelevels are measured using Abbott VP™ and VP Super System® Autoanalyzer(Abbott Laboratories, Irving, Tex.), using the A-Gent™ Triglycerides,Cholesterol and Glucose Test reagent systems, and a lactate kit fromSigma, respectively. The plasma insulin, triglycerides, totalcholesterol and lactate lowering activity of a growth hormone releasingpeptide (GHRP) or GHRP mimetic such as a compound of Formula I, aredetermined by statistical analysis (unpaired t-test) with thevehicle-treated control group.

[0118] The (L)-(+)-tartaric acid salt of the compound of Formula I canbe administered by oral, parenteral (e.g., intramuscular,intraperitoneal, intravenous or subcutaneous injection, or implant),nasal, vaginal, rectal, sublingual, or topical routes of administrationand can be formulated with pharmaceutically acceptable carriers toprovide dosage forms appropriate for each route of administration.

[0119] Solid dosage forms for oral administration include capsules,tablets, pills, powders and granules. In such solid dosage forms, the(L)-(+)-tartaric acid salt of the compound of Formula I is admixed withat least one inert pharmaceutically acceptable carrier such as sucrose,lactose, or starch. Such dosage forms can also comprise, as is normalpractice, additional substances other than such inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets and pills, the dosage forms may also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings.

[0120] Liquid dosage forms for oral administration includepharmaceutically acceptable emulsions, solutions, suspensions, syrups,the elixirs containing inert diluents commonly used in the art, such aswater. Besides such inert diluents, compositions can also includeadjuvants, such as wetting agents, emulsifying and suspending agents,and sweetening, flavoring and perfuming agents.

[0121] Preparations according to this invention for parenteraladministration include sterile aqueous or non-aqueous solutions,suspensions, or emulsions. Examples of non-aqueous solvents or vehiclesare propylene glycol, polyethylene glycol, vegetable oils, such as oliveoil and corn oil, gelatin, and injectable organic esters such as ethyloleate. Such dosage forms may also contain adjuvants such as preserving,wetting, emulsifying, and dispersing agents. They may be sterilized by,for example, filtration through a bacteria-retaining filter, byincorporating sterilizing agents into the compositions, by irradiatingthe compositions, or by heating the compositions. They can also bemanufactured in the form of sterile solid compositions which can bedissolved in sterile water, or some other sterile injectable mediumimmediately before use.

[0122] Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance,excipients such as coca butter or a suppository wax.

[0123] Compositions for nasal or sublingual administration are alsoprepared with standard excipients well known in the art.

[0124] The dosage of the (L)-(+)-tartaric acid salt of the compound ofFormula I in the compositions of this invention may be varied; however,it is necessary that the amount thereof be such that a suitable dosageform is obtained. The selected dosage depends upon the desiredtherapeutic effect, on the route of administration, and on the durationof the treatment. Generally, dosage levels of between 0.0001 to 100mg/kg of body weight daily are administered to humans and other animals,e.g., mammals, to obtain effective release of growth hormone.

[0125] A preferred dosage range is 0.01 to 5.0 mg/kg of body weightdaily which can be administered as a single dose or divided intomultiple doses.

[0126] The following scheme illustrates the synthesis of the(L)-(+)-tartaric acid salt of the compound of Formula I. The symbol *″indicates a stereochemical center. In the scheme “Prt” is used toindicate any suitable amine protecting group that will be known to thoseskilled in the art.

[0127] The following describes the steps of the reactions illustrated inthe foregoing scheme. In the following description, the amine protectinggroup Prt is illustrated with the preferred amine protecting group BOC.

[0128] Step a: To a solution of compound A in a reaction inert polaraprotic solvent such as acetone, methyl ethyl ketone or preferably DMF(dimethylformamide) at about 0° C. to room temperature, preferably 0°C., is added picolyl chloride hydrochloride, a carbonate such as Li₂CO₃,CsCO₃ or preferably potassium carbonate and potassium iodide ortetrabutylammonium iodide. After stirring at about −20° C. to about 70°C., preferably 0° C. for about 2 to 16 hours, preferably for about 2hours, the ice bath is removed and DABCO (1,4-diazobicyclo[2.2.2]octane)is added. The reaction mixture is stirred for about 15-30 min. andpoured into a mixture of water and a non-polar organic solvent such astoluene, diethyl ether or preferably IPE (isopropyl ether). The organiclayer is separated and worked-up using standard methods known in the artto yield compound B.

[0129] Step b: A 70% aqueous solution of CF₃CH₂NHNH₂ is used as anaqueous solution in ethanol, water or toluene, preferably the 70%aqueous solution of CF₃CH₂NHNH₂ is extracted with toluene. To a solutionof compound B in an organic solvent such as ethanol or preferablytoluene, is first added the toluene extracts containing the anhydrous2,2,2-trifluoroethyl hydrazine, followed by acetic acid. The reactionmixture is heated at about 60°-110° C., preferably 70 ° C., for about 30minutes to 12 hours, preferably 2 hours. The reaction mixture is cooledto room temperature and neutralized with an aqueous base such as NaHCO₃.The organic layer is separated and worked-up using standard methodsknown in the art to yield compound C.

[0130] Step c: An acid such as HCI in IPE or ethanol, triflic acid or analkyl sulfonic acid such as methanesulfonic acid is added to a solutionof compound C in a reaction inert organic solvent such as EtOH, IPE orpreferably CH₂Cl₂. The mixture is stirred for about 1-2 hours, thencooled to about 0° C. to room temp., preferably 0° C., and then an aminebase, such as triethylamine, or NH₄OH is added to the mixture. Themixture is allowed to warm to room temperature, diluted with additionalorganic solvent and worked-up using standard methods known in the art toyield compound D.

[0131] Step d: (D)- or (L)-Tartardc acid, preferably (D)-tararic acid,is added to Compound D in acetone/water (about 8:1 to about 9:1) atabout room temperature. The mixture is stirred at room temperature forabout 15 minutes to overnight, preferably overnight, the solid isfiltered, collected and washed with cold acetone, to yield the compoundof formula E, preferably compound E is the (D)-tartrate of a singleenantiomer.

[0132] Step e: To a solution of N-BOC-serine, preferablyN-BOC-(D)-serine, (compound F) in THFIDMF (about 1:1 to about 2:1) atabout 0° C. is added n-BuLi or a potassium tert-butoxide solution. Thereaction mixture is stirred at about 0° C. for about 10-30 min.preferably 20 min., then 2,4difluorobenzyi bromide is added. Afterwarming to room temperature and stirring for about 6-24 hours, thereaction mixture is concentrated in vacuo to remove the THF and anaqueous acid such as 1 N HCl is added to adjust the mixture to pH ofabout 3. The reaction mixture is then partitioned between water and anorganic solvent such as CH₂Cl₂ or IPE. The organic solution is worked-upusing standard methods known in the art to yield compound G, preferablyhaving the R-configuration at the stereocenter, also known as the(D)-enantiomer.

[0133] Step f: To a solution of compound G in an organic solvent such asTHF, CH₂Cl₂, IPE or a mixture thereof, preferably CH₂Cl₂/IPE (about1:1), is added an alkyl sulfonic acid such as methanesulfonic acid. Thesolid is filtered and washed with a CH₂Cl₂/IPE mixture (1:1) to affordcompound H, preferably having the R-configuration at the stereocenter,also known as the (D)-enantiomer.

[0134] Step g: To a solution of compound H in THF/water (about 4:1) isadded 2-tert-butoxycarbonylamino-2-methyl-propionicacid-2,5-dioxo-pyrrolidin-1-yl ester and an alkyl amine such astriethylamine. The reaction mixture is stirred at room temperature forabout 1-24 hours and quenched with an aqueous acid such as 10% aqueouscitric acid solution. The mixture is partitioned with an organic solventsuch as ethyl acetate and the organic layer is separated and worked-upusing standard methods known in the art to yield compound X, preferablyhaving the R-configuration at the stereocenter also known as the(D)-enantiomer. Compound X can be an acid, alkyl ester or acid halide (Xis OH, —O(C₁-C₄)alkyl or halo), the acid is preferred.

[0135] Step h: (a) Compound E, preferably the (D)-tartrate of a singleenantiomer, is added at about −35° to 0° C., preferably at about −6° C.to ethyl acetate. The solution is cooled to about −30 to −50° C., thenan alkyl amine such as triethylamine is added. The reaction mixture isstirred for about 30-90 min. at a temperature between about −78° C. andabout −20° C., and filtered to give a solution of the free base ofcompound E.

[0136] (b) When X in compound X is OH, compound X, preferably having theR-configuration at the stereocenter, is added at about −78° C. to −20°C., preferably −35° C. to a reaction inert organic solvent such as ethylacetate solution containing the free base of compound E from step h(a),an alkyl amine such as triethylamine and PPAA (1-propane phosphonic acidcyclic anhydride) (50% in ethyl acetate). The reaction mixture isstirred for about 1-24 hours, and worked-up using standard methods knownin the art to yield compound J, preferably having the absolute andrelative 3a-(R), 1-(R) configuration.

[0137] When X in compound X is Cl, compound X is added at about −78° C.to a reaction inert solvent such as dichioromethane solution containingthe free base of compound E and an alkyl amine such as triethylamine.The reaction mixture is stirred for about 1-24 hours at about 0-30° C.and then worked up using standard methods known in the art to yieldcompound J, preferably having the absolute and relative 3a-(R), 1-(R)configuration.

[0138] When X in compound X is —O(C₁-C₄)alkyl, where methyl ispreferred, compound X is added to a solution of the free or conjugatebase of E (the conjugate base of compound E (—NM where M=Li, Na, K, Mgor Al, preferably aluminum) is prepared by reacting the free amine basewith the appropriate reagent (i.e. M=Li, butyl lithium or LDA, M=Na, NaHor NaN(SiMe₃)₂ or M=K, KH or KN(SiMe₃)₂, or M=Mg, any alkyl Grignardreagent, preferably diethyl magnesium bromide, or M=Al any trialkylaluminum reagent, preferably trimethyl aluminum)), preferably aluminum,in a reaction inert solvent such as dichloromethane and the resultingreaction mixture is stirred for about 1-24 hours at about −20-110° C.and worked-up using standard methods known in the art to yield compoundJ, preferably having the absolute and relative 3a-(R), 1-(R)configuration.

[0139] Step i: An acid such as HCl in EtOH, methanesulfonic acid ortriflic acid in CH₂Cl₂ is added at about 0° C. to room temperature tocompound J in CH₂Cl₂, IPE or THF. The mixture is stirred for about 40minutes to about 4 hours at room temperature, then a saturated aqueousbase such as NaHCO₃ is added until the solution is at neutral pH. Theorganic layer is separated and worked-up using standard methods known inthe art to yield compound K, preferably having the absolute and relative3a-(R), 1-(R) configuration.

[0140] Step j: To a solution of compound K in an alcohol preferablymethanol is added L-(+) tartaric acid. The reaction mixture is stirredfor about 1-12 hours, filtered and concentrated. The crude residue isdiluted with an organic solvent such as ethyl acetate, heated and slowlyallowed to cool to room temperature. The solid is filtered and dried togive the L-(+) tartaric acid salt of the compound of Formula I as whitecrystals, preferably having the absolute and relative 3a-(R), 1-(R)configuration.

[0141] The following example is provided for the purpose of furtherillustration only and is not intended to be a limitation on thedisclosed invention.

[0142] Silica gel was used for column chromatography. Melting pointswere taken on a Buchi 510 apparatus and are uncorrected. Proton NMRspectra were recorded on a Varian XL-300, Bruker AC-300, Varian Unity400 or Bruker AC-250 at 25° C. Chemical shifts are expressed in partsper million down field from trimethylsilane.

EXAMPLE 12-Amino-N-{1-(2,4-difluoro-benzyloxymethyl)-2-oxo-2-[3-oxo-3a-pyridin-2-ylmethyl-2-(2,2.2-trifluoro-ethyl)-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5-yl]-ethyl}-2-methyl-propionamideL-(+) tartrate

[0143] A. 4Oxo-3-pyridin-2-ylmethyl-piperidine-1,3-dicarboxylic acid1-tert-butyi ester 3-ethyl ester

[0144] To a solution of 4-oxo-piperidine-1,3-dicarboxylic acid1-tert-butyl ester 3-ethyl ester (10.34 g, 38.2 mmol) in DMF (40 mL) atabout 0° C. was added picolyl chloride hydrochloride (5.7 g, 34.7 mmol),potassium carbonate (14.4 g, 104.1 mmol) and potassium iodide (5.76 g,34.7 mmol). After stirring at about 0° C. for about 2 hours, the icebath was removed and DABCO (973 mg, 8.68 mmol) was added. The reactionmixture was stirred for about 30 min. and poured into a mixture of waterand IPE. The organic layer was separated and washed with saturatedaqueous NaHCO₃ and saturated aqueous NaCl, dried over Na₂SO₄ andconcentrated in vacuo. The crude residue was crystallized from hexanesto give a white solid (8.19 g, yield 65%). ¹H-NMR (CDCl₃) δ 1.17 (t,3H), 1.48 (s, 9H), 1.55 (s, 2H), 2.61 (m, 1H), 2.71 (m, 1H), 3.31-3.50(m, 3H), 4.11 (d, 2H), 4.49 (d, 1H), 7.06 (brs, 1H), 7.17 (d, 1H), 7.54(m, 1H), 8.40 (s, 1H).

[0145] B.3-Oxo-3a-pyridin-2-ylmethyl-2-(2,2,2-trifluoro-ethyl)-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridine-5-carboxylicacid tert-butyl ester

[0146] A 70% aqueous solution of CF₃CH₂NHNH₂ (325 mL, 1.986 mol)(obtained from Aldrich) was extracted with toluene (3×1200 mL). To asolution of the product made according to step A (600 g, 1.655 mol) intoluene (900 mL) was first added the combined toluene extractscontaining the anhydrous 2,2,2-trifluoroethyl hydrazine, followed byacetic acid (121.4 g, 1.986 mol). The reaction mixture was heated atabout 70° C. for about 2 hours , then another toluene extraction of 70%aqueous 2,2,2-trifluoroethyl hydrazine (50 g) was added. The reactionmixture was heated at about 80° C. for about 3.5 hours, cooled to roomtemperature and diluted with saturated aqueous NaHCO₃ (2 L). The toluenelayer was separated and washed with saturated aqueous NaCl, dried overNa₂SO₄ and concentrated in vacuo to give an oil (754.8 g).Crystallization from methanol/water afforded the desired product as awhite solid (609.5 g). ¹H-NMR (CDCl₃) δ 1.50 (s, 9H), 2.53 (d, 1H), 2.70(br s, 2H), 2.88 (br s, 1H), 3.31 (m, 2H), 3.97 (m, 1H), 4.19 (m, 1H),4.46 (br s, 1H), 4.63 (br s, 1H), 7.06 (m, 2H), 7.51 (m, 1H), 8.34 (m,1H).

[0147] C.3a-Pyridin-2-ylmethyl-2-(2,2,2-trifluoroethyl)-2.3a,4,5,6,7-hexahydro-pyrazolo[4,3-c]pyridin-3-one

[0148] Methanesulfonic acid (11.6 g, 121 mmol) was added dropwise to asolution of the product from step B (10 g, 24.2 mmol) in CH₂Cl₂ (100 mL)over about 30 minutes. The reaction mixture was stirred for about 1hour, then cooled to about 0° C., and then triethylamine (18.6 mL, 133.1mmol) was added through an addition funnel. The mixture was allowed towarm to room temperature over about 1 hour, diluted with additionalCH₂Cl₂ and washed with saturated aqueous NaCl, dried over Na₂SO₄,filtered and concentrated in vacuo to afford the product as a whitesolid (7.2 g). ¹H-NMR (CDCl₃) δ: 2.51-2.72 (m, 4H), 3.35 (m, 2H), 3.49(m, 2H), 4.03 (m, 1H), 4.25 (m, 1H), 7.08 (d, 2H), 7.51 (t, 1H), 8.37(d, 1H).

[0149] D.3a-Pyridin-2-ylmethyl-2-(2,2,2-trifluoroethyl)-2,3a.4,5,6,7-hexahydro-pyrazolo[4,3-c]pyridin-3-one(D)-tartrate

[0150] In a dry and nitrogen purged 5 L round bottom flask equipped witha mechanical stirrer, D-(-) tartaric acid (129 g, 0.86 mol) was added tothe compound made according to step C (243 g, 0.78 mol) in acetone/water(9:1, 2430 mL) at about 17° C. The mixture was stirred at roomtemperature overnight, filtered, the solid was collected and washed withcold acetone and dried under vacuum. The product was obtained as ayellow solid (284 g, yield 78.8%).

[0151] E.2-tert-Butoxycarbonylamino-3-(2,4-difluoro-benzyloxy)-propionic acid

[0152] To a solution of N-Boc-(D)-serine (452 g, 2.2026 mol) in amixture of THF (7 L) and DMF (3 L) at about 0° C. was added potassiumtert-butoxide solution (515.8 g, 4.5963 mol). The reaction mixture wasstirred at about 0° C. for about 30 min., then 2,4-difluorobenzylbromide (456.5 g, 2.2051 mol) was added. After warming to roomtemperature, the reaction mixture was concentrated in vacuo to removethe THF. Partitioned the reaction mixture between 4.5 L H₂O and 4.5 LIPE. Separated the layers and adjusted the pH of the aqueous layer with1 N HCl to about 3. The aqueous layer was extracted twice with 4 L eachof IPE. The organic solution was dried over Na₂SO₄, and concentrated invacuo to yield a yellow waxy solid (518.0 g, yield: 70.9%). ¹H-NMR(CDCl₃) δ 1.44 (s, 9H), 3.73 (m, 1H), 3.94 (d, 1H), 4.44 (br s, 1H),4.54 (s, 2H), 5.34 (m, 1H), 6.78 (m, 1H), 6.84 (m, 1H), 730 (m, 1H).

[0153] F. 2-Amino-3-(2,4-difluoro-benzyloxy)-propionic acid,methanesulfonic acid salt

[0154] To a solution of the product from step E (1.19 g, 3.59 mmol) inCH₂Cl₂/IPE (1:1, 12 mL) was added methanesulfonic acid (1.72 g, 17.95mmol) through a syringe over about 10 minutes. A solid immediatelyprecipitated out of solution. After about 1 hour, the solid was filteredand washed with a CH₂Cl₂/IPE mixture (1:1) to afford 939 mg of product(yield 80%).

[0155] G.2-(2-tert-Butoxycarbonylamino-2-methyl-propionylamino)-3-(2,4-difluoro-benzyloxy)-propionicacid

[0156] To a solution of the product from step F (520 mg, 1.46 mmol) inTHF/water (4:1, 10 mL) was added2-tert-butoxycarbonylamino-2-methyl-propionicacid-2,5-dioxo-pyrrolidin-1-yl ester (438 mg, 1.46 mmol) andtriethylamine (369 mg, 3.65 mmol). The reaction mixture was stirred atroom temperature for about 1 hour and quenched with a 10% aqueous citricacid solution (10 mL). After about 15 min., ethyl acetate (50 mL) wasadded and the organic layer was separated and washed with saturatedaqueous NaCl, dried over Na₂SO₄ and concentrated in vacuo to give a foam(534.1 mg, yield 88%). ¹H-NMR (CD₃OD): δ 1.38 (br s, 15H), 3.77 (d, 1H),3.92 (d, 1H), 4.52 (m, 3H), 6.92 (m, 1H), 7.41 (m, 1H), 7.58 (d, 1H).

[0157] H.(1-{2,4-Difluoro-benzyloxymethyl-2-oxo-2-[3-oxo-3a-pyridin-2-ylmethyl-2-(2,2,2-trifluoro-ethyl)-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5-yl]-ethylcarbamoyl}-1-methyl-ethyl)-carbamicacid tert-butyl ester

[0158] (a) To the compound made according to step D (517 g, 1.12 mol)was added at about −6° C. to ethyl acetate (5170 mL) in a dry andnitrogen purged 12 L round bottom flask equipped with a mechanicalstirrer. The solution was cooled to about −40° C., then triethylamine(398 mL, 2.86 mol) was added over about 45 minutes. The reaction mixturewas stirred for about 90 min. at a temperature between about −50° C. andabout −40° C., filtered into a 22 L round bottom flask purged withnitrogen and washed with ethyl acetate (2068 mL, pre-cooled to about−50° C.) to give the free base as a white solid.

[0159] (b) The compound made according to step G (425 g, 1.02 mol ) wasadded at about −30° C. to an ethyl acetate solution containing theproduct from step H (a), triethylamine (654 mL, 4.69 mol) and PPAA(1-propanephosphonic acid cyclic anhydride) (50% in ethyl acetate, 916mL, 1.53 mol). The reaction mixture was stirred for about 1 hour, washedwith water and saturated aqueous NaCl, dried over Na₂SO₄ andconcentrated in vacuo to give the product as an oil (636 g, yield:87.8%).

[0160] I.2-Amino-N-{1-(2,4-difluoro-benzyloxymethyl)-2-oxo-2-[3-oxo-3a-pyridin-2-ylmethyl-2-(2,2,2-trifluoro-ethyl)-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5-yl]-ethyl}-2-methyl-propionamide

[0161] Methanesulfonic acid (258.3 mL, 3.98 mol) was added dropwise atabout 15° C. over about 55 minutes to the product from step H (566 g,0.796 mol) in CH₂Cl₂ (11,320 mL) in a dry and nitrogen purged 22 L roundbottom flask equipped with a mechanical stirrer. The mixture was stirredfor about 40 minutes at about 20° C., then saturated aqueous NaHCO₃(8,490 mL) was added until the pH was about 7.8. The organic layer wasseparated, washed with water and saturated aqueous NaCl, dried overNa₂SO₄, and concentrated in vacuo to afford an oily product (388.8 g,yield 80%).

[0162] J.2-Amino-N-{1-(2,4-difluoro-benzytoxymethyl)-2-oxo-2-[3-oxo-3a-pyridin-2-ylmethyl-2-(2,2,2-trifluoro-ethyl)-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5-yl]-ethyl}-2-methyl-propionamideL-(+) tartrate

[0163] To a solution of the product from step I (370 g, 0.6 mol) inmethanol (4,070 mL) in a 12 L round bottom flask equipped with amechanical stirrer was added L-(+) tartaric acid (90 g, 0.6 mol). Thereaction mixture was stirred for about 90 min. at about 22° C., filteredand concentrated. The crude residue was diluted with ethyl acetate(4,560 mL), heated at about 70° C. and slowly allowed to cool to roomtemperature over about 17 hours. The solid was filtered and dried togive white crystals, mp 188-189° C. (348.46 g, yield 76%). ¹H NMR (MeOH,d4) δ: 8.28 (d, 1H), 7.59 (t, 1H), 7.41-7.39 (m, 1H), 7.18-7.13 (m, 1H),6.92 (t, 1H), 5.2 (t, 1H), 4.56 (bs, 3H), 4.36 (s, 2H), 4.31-4.25 (m,1H), 4.13-4.06 (m, 1H), 3.78 (d, 2H), 3.21 (t, 1H), 3.18-2.96 (m, 2H),2.65-2.55 (m, 2H), 1.57 (d, 6H). MS: MH+611. [a]⁵⁸⁹+22.03 (c=11.9,MeOH).

What is claimed is:
 1. The (L)-(+)-tartaric acid salt of the compound ofFormula I


2. The (L)-(+)-tartaric acid salt of the compound of formula I accordingto claim 1 wherein the stereochemical configuration is 3a-S, 1-R.
 3. The(L)-(+)-tartaric acid salt of the compound of formula I according toclaim 1 wherein the stereochemical configuration is 3a-S, 1-S.
 4. The(L)-(+)-tartaric acid salt of the compound of formula I according toclaim 1 wherein the stereochemical configuration is 3a-R, 1-S.
 5. The(L)-(+)-tartaric acid salt of the compound of formula I according toclaim 1 wherein the stereochemical configuration is 3a-R, 1-R.
 6. Aprocess for the preparation of the (D)-tartaric acid or the (L)-tartaricacid salt of the compound of formula (E),

which comprises reacting the compound of formula (D),

with (D)-tartaric acid or (L)-tartaric acid in about 8:1 to about 9:1mixture of acetone:water at a temperature between about 0° C. to roomtemperature.
 7. A process according to claim 6 wherein (D)-tartaric acidis reacted with the compound of formula (D) and the compound of formula(E) has the R-configuration.
 8. A process for the preparation of thecompound of formula (J),

which comprises reacting the compound of formula (E),

with the compound of formula (X),

where Prt is an amine protecting group and X is OH, —O(C₁-C₄)alkyl orhalo, in the presence of an organic base and a peptide coupling reagentat a temperature between about −78° C. to about −20° C.
 9. A processaccording to claim 8 where the peptide coupling reagent is 1-propanephosphonic acid cyclic anhydride and the compound of formula X has theR-configuration and the compound of formula E has the R-configuration.10. A process according to claim 9 wherein Prt is tert-butoxycarbonyl.11. A process for the preparation of the (L)-(+)-tartaric acid salt ofthe compound of formula I,

which comprises reacting the compound of formula (E),

with the compound of formula (X),

where Prt is an amine protecting group and X is OH, —O(C₁-C₄)alkyl orhalo, in the presence of an organic base and a peptide coupling reagentat a temperature between about −78° C. to about −20° C., to yield thecompound of formula (J),

deprotecting the compound of formula (J) under appropriate deprotectingconditions to yield the compound of formula (K),

reacting the compound of formula (K) with (L)-(+)-tartaric acid in areaction inert solvent to yield the (L)-(+)-tartaric acid salt of thecompound of formula I.
 12. A process according to claim 11 where Prt istert-butoxycarbonyl.
 13. A process according to claim 12 where thepeptide coupling reagent is 1-propane phosphonic acid cyclic anhydrideand the compound of formula I has the absolute and relativeconfiguration 3a-(R), 1-(R).
 14. A method for increasing levels ofendogenous growth hormone in a human or other animal which comprisesadministering to such human or animal an effective amount of the(L)-(+)-tartaric acid salt of the compound of formula I according toclaim 1 .
 15. A pharmaceutical composition which comprises apharmaceutically-acceptable carrier and an amount of the(L)-(+)-tartaric acid salt of the compound of formula I according toclaim 1 .
 16. A pharmaceutical composition useful for increasing theendogenous production or release of growth hormone in a human or otheranimal which comprises a pharmaceutically acceptable carrier, aneffective amount of the (L)-(+)-tartaric acid salt of the compound offormula I according to claim 1 and a growth hormone secretagogueselected from the group consisting of GHRP-6, Hexarelin, GHRP-1, growthhormone releasing factor (GRF), IGF-1, IGF-2 and B-HT920 or an analogthereof.
 17. A method for treating or preventing osteoporosis whichcomprises administering to a human or other animal in need of suchtreatment or prevention an amount of the (L)-(+)-tartaric acid salt ofthe compound of formula I according to claim 1 which is effective intreating or preventing osteoporosis.
 18. A method for treating orpreventing diseases or conditions which may be treated or prevented bygrowth hormone which comprises administering to a human or other animalin need of such treatment or prevention an amount of the(L)-(+)-tartaric acid salt of the compound of formula I according toclaim 1 which is effective in promoting release of endogenous growthhormone.
 19. A method according to claim 18 wherein the disease orcondition is congestive heart failure, obesity or frailty associatedwith aging.
 20. A method according to claim 19 wherein the disease orcondition is congestive heart failure.
 21. A method according to claim19 wherein the disease or condition is frailty associated with aging.22. A method for accelerating bone fracture repair, attenuating proteincatabolic response after a major operation, reducing cachexia andprotein toss due to chronic illness, accelerating wound healing, oraccelerating the recovery of bum patients or patients having undergonemajor surgery, which method comprises administering to a mammal in needof such treatment an amount of the (L)-(+)-tartaric acid salt of thecompound of formula I according to claim 1 which is effective inpromoting release of endogenous growth hormone.
 23. A method accordingto claim 22 wherein the method is for accelerating the recovery ofpatients having undergone major surgery.
 24. A method according to claim22 wherein the method is for accelerating bone fracture repair.
 25. Amethod for improving muscle strength, mobility, maintenance of skinthickness, metabolic homeostasis or renal homeostasis, which methodcomprises administering to a human or other animal in need of suchtreatment an amount of the (L)-(+)-tartaric acid salt of the compound offormula I according to claim 1 which is effective in promoting releaseof endogenous growth hormone.
 26. A method for the treatment orprevention of osteoporosis which comprises administering to a human orother animal with osteoporosis effective amounts of a bisphosphonatecompound and the (L)-(+)-tartaric acid salt of the compound of formula Iaccording to claim 1 .
 27. A method for the treatment of osteoporosisaccording to claim 26 wherein the bisphosphonate compound isibandronate.
 28. A method for the treatment of osteoporosis according toclaim 26 wherein the bisphosphonate compound is alendronate.
 29. Amethod for the treatment or prevention of osteoporosis which comprisesadministering to a human or other animal with osteoporosis effectiveamounts of estrogen or Premarin® and the (L)-(+)-tartarc acid salt ofthe compound of formula I according to claim 1 and, optionally,progesterone.
 30. A method for the treatment of osteoporosis whichcomprises administering to a human or other animal with osteoporosiseffective amounts of calcitonin and the (L)-(+)-tartaric acid salt ofthe compound of formula I according to claim 1 .
 31. A method toincrease IGF-i levels in a human or other animal deficient in IGF-1which comprises administering to a human or other animal with IGF-1deficiency an effective amount of the (L)-(+)-tartaric acid salt of thecompound of formula I according to claim 1 .
 32. A method for thetreatment of osteoporosis which comprises administering to a human orother animal with osteoporosis effective amounts of an estrogen agonistor antagonist and the of.the (L)-(+)-tartaric acid salt of the compoundof formula I according to claim 1 .
 33. A method according to claim 32wherein the estrogen agonist or antagonist is tamoxifen, droloxifene,raloxifene or idoxifene.
 34. A method according to claim 32 wherein theestrogen agonist or antagonist iscis-6-(4-fluoro-phenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalene-2-ol;(-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalene-2-ol;cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalene-2-ol;cis-1-[6′-pyrrolodinoethoxy-3′-pyridyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahydro-naphthalene;1-(4′-pyrrolidinoethoxyphenyl)-2-(4″-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;cis-6-(4-hydroxyphenyl)-5-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalene-2-ol;or1-(4′-pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydro-isoquinoline.35. A method for increasing muscle mass, which method comprisesadministering to a human or other animal in need of such treatment aneffective amount of the (L)-(+)-tartaric acid salt of the compound offormula I according to claim 1 .
 36. A method for promoting growth ingrowth hormone deficient children which comprises administering to agrowth hormone deficient child an effective amount of the(L)-(+)-tartaric acid salt of the compound of formula I according toclaim 1 .
 37. A method for treating insulin resistance in a mammal,which 15 comprises administering to said mammal an effective amount ofthe (L)-(+)-tartaric acid salt of the compound of formula I according toclaim 1 .
 38. A method according to claim 37 wherein the conditionassociated with insulin resistance is type I diabetes, type II diabetes,hyperglycemia, impaired glucose tolerance or an insulin resistantsyndrome.
 39. A method according to claim 37 wherein the conditionassociated with insulin resistance is associated with obesity or oldage.
 40. A method for increasing levels of endogenous growth hormone,which comprises administering to a human or other animal in need thereofeffective amounts of a functional somatostatin antagonist and the(L)-(+)-tartaric acid salt of the compound of formula I according toclaim 1 .
 41. A method according to claim 40 wherein the functionalsomatostatin antagonist is an alpha-2 adrenergic agonist.
 42. A methodof treating or preventing congestive heart failure, obesity or frailtyassociated with aging, which comprises administering to a human or otheranimal in need thereof effective amounts of a functional somatostatinantagonist and the of the (L)-(+)-tartaric acid salt of the compound offormula I according to claim 1 .
 43. The R,S-enantiomeric mixture, theR-enantiomer or the S-enantiomer of the compound of the formula


44. The (D)-tartaric acid or the (L)-tartaric acid salt of the compoundaccording to claim 43 .
 45. The 3a-(R,S), 1-(R) diastereomeric mixture,the 3a-(R), 1-(R) diastereomer or the 3a-(S), 1-(R) diastereomer of thecompound of the formula

where Prt is an amine protecting group selected from the groupconsisting of t-BOC, FMOC and CBZ.
 46. The R,S-enantiomeric mixture, theR-enantiomer or the S-enantiomer of the compound of the formula


47. The R,S-enantiomeric mixture, the R-enantiomer or the S-enantiomerof the compound of the formula

where X is OH, —O(C₁-C₄)alkl or halo and Prt is an amine protectinggroup.
 48. The compound according to claim 47 where X is OH, Prt is BOCand the stereocenter is in the R-configuration.
 49. A method of treatingsleep disorders in a mammal suffering from sleep disorders comprisingadministering to said mammal an effective amount of the (L)-(+)-tartaricacid salt of the compound of formula I according to claim 1 .